US20230265512A1 - Method for diagnosing down's syndrome by using down's syndrome-specific epigenetic marker - Google Patents

Method for diagnosing down's syndrome by using down's syndrome-specific epigenetic marker Download PDF

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US20230265512A1
US20230265512A1 US18/003,609 US202118003609A US2023265512A1 US 20230265512 A1 US20230265512 A1 US 20230265512A1 US 202118003609 A US202118003609 A US 202118003609A US 2023265512 A1 US2023265512 A1 US 2023265512A1
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Hyun Mee RYU
Ji Hyae LIM
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Sungkwang Medical Foundation
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Definitions

  • the present disclosure relates to a Down syndrome-specific epigenetic marker and a method of providing information for diagnosing Down syndrome and a method of diagnosing Down syndrome.
  • Down syndrome a type of disease caused by the most common chromosomal abnormality, the embryo survives.
  • WHO World Health Organization
  • Down syndrome the most common genetic cause of developmental disabilities, has an estimated prevalence of 1 to 100 in 1,100 worldwide.
  • Down syndrome exhibits more than 80 clinical features, including mental retardation, facial features, poor muscle development, and short stature, and is related to an increased risk of congenital heart disease, diabetes, leukemia, and other diseases.
  • a technique in the art for diagnosing Down syndrome is a method of analyzing the quantitative difference of cfDNA, a non-cellular DNA fragment present in plasma of pregnant women (next generation sequencing (NGS)-based non-invasive prenatal testing (NIPT)).
  • NGS next generation sequencing
  • NIPT non-invasive prenatal testing
  • the present inventors have completed the present disclosure, which may effectively test for Down syndrome through simple measurement of a methylation level of the genome, without the need to utilize existing expensive test equipment or special algorithms.
  • An aspect provides a method of providing information for diagnosing Down syndrome, the method including: measuring a methylation level of a Down syndrome biomarker in a biological sample separated from a subject, wherein the biomarker is a first biomarker present on chromosome 21, a second biomarker present on a chromosome other than chromosome 21, or a combination thereof; comparing the measured methylation level of the biomarker with a methylation level of the first biomarker and the second biomarker in the biological sample separated from a normal control group; and determining a presence or a risk of Down syndrome by comparing the methylation levels.
  • the method including: measuring a methylation level of a Down syndrome biomarker in a biological sample separated from a subject, wherein the biomarker is a first biomarker present on chromosome 21, a second biomarker present on a chromosome other than chromosome 21, or a combination thereof; comparing the measured methylation level of the biomarker with a methylation level of the first biomarker and the second biomarker in the biological sample separated from a normal control group; and determining a presence or a risk of Down syndrome by comparing the methylation levels.
  • Down syndrome refers to a kind of congenital genetic disease, in which one more full or partial copy of chromosome 21 is present than normal. Down syndrome causes delayed physical development and may be accompanied by facial deformities and intellectual disabilities. Most of the chromosomes of a patient with a Down syndrome, which are inherited from parents, are normal, but there may be one more copy of chromosome 21 due to nondisjunction of reproductive cells during pregnancy. The incidence rate is 0.1% in 20-year-old mothers, but increases rapidly to 4% in 45-year-old mothers, and the incidence rate shows a tendency to increase with the mother's age. Down syndrome may be confirmed by a prenatal diagnosis of the fetus during pregnancy, and may be determined through direct genetic testing after childbirth.
  • diagnosis refers to confirming a presence or characteristics of a pathological state, and may include determining whether or not Down syndrome has occurred or is likely to occur.
  • methylation may mean that a methyl group is added to the 5th carbon of a cytosine residue of DNA, and may mean that a methyl group is attached to a base constituting DNA.
  • an occurrence of methylation means an occurrence of methylation at the fifth carbon of a cytosine residue of a specific CpG site of a specific gene.
  • 5-methylcytosine In genomic DNA of mammalian cells, there is a fifth base called 5-methylcytosine (5-mC) with a methyl group attached to the fifth carbon of a cytosine ring, in addition to A, C, G, and T. Methylation of 5-methylcytosine occurs only at C of CG dinucleotide (5′-mCG-3′) called CpG, and methylation of CpG inhibits expression of alu or transposon and genomic repetitive sequences. In addition, since 5-mC of CpG is easy to be naturally deaminated to become thymine (T), CpG is a site where most epigenetic changes occur frequently in mammalian cells.
  • 5-mC of CpG is easy to be naturally deaminated to become thymine (T)
  • T thymine
  • the term “measurement of a methylation level”, used herein, may include measuring a level of methylation of Down syndrome-related gene biomarkers in a biological sample in order to diagnose Down syndrome.
  • the measurement of a methylation level is to measure methylation levels of CpG sites, and any method known in the art for measuring a methylation level may be used without limitation, but methylation-specific PCR, for example, methylation-specific polymerase chain reaction (MSP), real-time methylation-specific polymerase chain reaction, PCR using binding proteins specific for methylated DNA, quantitative PCR, PCR using methylation-specific specific peptide nucleic acid (PNA), or melting curve analysis may be used.
  • MSP methylation-specific polymerase chain reaction
  • PNA methylation-specific specific peptide nucleic acid
  • a methylation level may be measured by methods such as DNA chip, pyrosequencing, bisulfite sequencing, and automatic sequencing such as methyl-capture sequencing (MC-S
  • DMC differentiated CpG site
  • DMR differentiated CpG region
  • the agent for measuring a methylation level may be an agent for confirming the presence or absence of methylation of a gene, and may be for measuring an amount of methylated genes.
  • the agent for measuring a methylation level may include, for example, a compound or methylation-specific restriction enzyme (MSRE) that modifies unmethylated cytosine bases, a primer specific for a methylated sequence of the gene, and a primer specific for an unmethylated sequence.
  • the compound that modifies the unmethylated cytosine base may be bisulfite, but is not limited thereto, and may be preferably sodium bisulfite. This method of detecting methylation of a promoter by modifying an unmethylated cytosine residue by using bisulfite is well known in the art.
  • the methylation-specific restriction enzyme refers to an enzyme that selectively cuts nucleic acids according to the methylation state of its restriction site. For restriction enzymes that specifically cleave when the restriction site is unmethylated or hemimethylated, cleavage will not occur or will occur with significantly reduced efficiency when the restriction site is methylated. For restriction enzymes that specifically cleave when the restriction site is methylated, cleavage will not occur or will occur with significantly reduced efficiency when the restriction site is unmethylated.
  • Another aspect provides a use of the first biomarker for diagnosing Down syndrome.
  • the first biomarker may be at least one selected from the group presented in Table 1 below.
  • CHODL chondrolectin gene
  • NCAM2 gene, used herein, refers to a gene encoding a neural cell adhesion molecule 2 protein. The gene is known to be associated with a prion disease.
  • CYYR1 gene refers to a gene encoding cysteine and tyrosine-rich protein 1 (CYYR1). The function of the protein encoded by the gene has not been specifically known.
  • GKIK1 refers to a gene encoding a glutamate receptor, ionotropic, kainate 1 (GRIK1) protein.
  • the gene encodes one of many subunits of an ionic glutamate receptor (GluR) that function as ligand-gated ion channels.
  • GluR ionic glutamate receptor
  • OLIG2 refers to a gene encoding an oligodendrocyte transcription factor protein. It is known that the expression of the gene is mainly restricted in the central nervous system, where the gene acts as both an anti-neurigenic and a neurigenic factor at different stages of development, and that the gene is mainly associated with brain tumors.
  • CLIC6 gene, used herein, refers to a gene encoding chloride intracellular channel protein 6. The gene is known to interact with the dopamine receptor D3.
  • SIM2 gene refers to a gene encoding a single-minded homolog 2 protein.
  • the protein encoded by the gene is known to play an important role in the development of the midline of the central nervous system as well as the construction of the face and head.
  • HLCS holocarboxylase synthetase protein
  • the protein encoded by the gene plays an important role in effectively using vitamin B (biotin) found in foods such as egg yolk and milk, and is involved in many important cellular functions including production and breakdown of proteins, fats, and carbohydrates.
  • MX2 gene, used herein, refers to a gene encoding interferon-induced GTP-binding protein Mx2. It is known that the protein encoded by the gene is up-regulated by interferon-alpha, but does not include the antiviral activity of the similar myxovirus resistance protein 1.
  • MX1 gene refers to a gene encoding interferon-induced GTP-binding protein Mx1. Interferon-induced Mx proteins are known to be associated with a specific antiviral state against influenza virus infection in mice.
  • TMPRSS2 transmembrane protease, serine 2 protein.
  • Serine proteases are known to be involved in many physiological and pathological processes, and the gene is known to be up-regulated by androgen hormones in prostate cancer cells and down-regulated in androgen-independent prostate cancer tissues. However, the specific biological function of the gene is unknown.
  • SLC37A1 gene refers to a gene encoding a glucose-6-phosphate exchanger SLC37A1 protein. Unlike a SLC37A4 protein, the protein encoded by the gene does not appear to be involved in blood sugar homeostasis, but is known to regulate phosphate levels in cow's milk and affect the amount of milk produced.
  • PDE9A gene refers to a gene encoding a high affinity cGMP-specific 3′,5′-cyclic phosphodiesterase 9A protein.
  • the protein encoded by the genes is known to play a role in signal transduction by regulating intracellular concentrations of cAMP and cGMP.
  • CBS gene refers to a gene encoding a cystathionine beta-synthase. Defects in the gene are known to cause cystathionine beta-synthase deficiency (CBSD), resulting in homocystinuria.
  • CRYAA gene refers to a gene encoding an alpha-crystallin A chain protein. Defects in the gene are known to cause autosomal dominant congenital cataract.
  • C21orf2 gene refers to a gene encoding cilia and flagella associated protein 410 (CFAP410).
  • the gene is known to be associated with retinal dystrophy and spondylometaphyseal dysplasia.
  • TRPM2 gene, used herein, refers to a gene encoding a transient receptor potential cation channel, subfamily M, member 2 protein. Although the physiological function of the gene is not precisely known, the gene has been reported to be involved in insulin secretion.
  • TSPEAR thrombospondin type laminin G domain and EAR repeats protein.
  • the gene is known to be related to hearing loss (deafness) and ectodermal dysplasia.
  • LINC00162 gene refers to a P38 inhibited cutaneous squamous cell carcinoma associated LincRNA (PICSAR) gene.
  • PICSAR cutaneous squamous cell carcinoma associated LincRNA
  • SSR4P1 gene, used herein, refers to signal sequence receptor subunit 4 pseudogene 1. The exact function of the gene is not known.
  • SLC19A1 gene refers to a gene encoding a folate transporter 1 protein. It is known that the protein encoded by the gene plays an important role in maintaining the concentration of folic acid in cells.
  • LOC100129027 gene refers to a PCBP3 antisense RNA 1 (PCBP3-AS1) gene. The specific biological function of the gene is unknown.
  • MCM3AP gene, used herein, refers to a gene encoding an 80 kDa MCM3-associated protein.
  • the protein encoded by the gene is an MCM3 binding protein, which is known to have a phosphorylation-dependent DNA-primase activity.
  • YBEY gene refers to a gene encoding a YbeY metalloendoribonuclease.
  • the gene is known to be associated with mesenteric lymphadenitis.
  • PRMT2 gene, used herein, refers to a gene encoding a protein arginine N-methyltransferase 2.
  • the protein encoded by the gene is known to interact with estrogen receptor alpha.
  • ITSN1 gene refers to a gene encoding an intersectin-1 protein.
  • the gene is known to be associated with vaccinia and schizophrenia 1.
  • Another aspect provides a use of the second biomarker for diagnosing Down syndrome.
  • the second biomarker may be at least one selected from the group presented in Table 2 below.
  • MXRA8 gene refers to a gene encoding a matrix remodeling associated 8 protein.
  • the gene or the protein encoded thereby has been known as a biomarker for diagnosing non-muscle invasive bladder cancer (Korean Patent Publication No. 10-2019-0089552).
  • MIB2 gene, used herein, refers to a gene encoding a mindbomb E3 ubiquitin-protein ligase 2 (MIB2).
  • MIB2 encoded by the gene interacts with actin proteins (alpha 1) and is known to inhibit melanoma invasion.
  • KIF26B gene refers to a gene encoding a kinesin family member 26B (KIF26B) protein.
  • the protein encoded by the gene is an intracellular motor protein that transports cell organelles along microtubules, and is essential for kidney development, and increased levels of the protein have been observed in some breast and colorectal cancers.
  • SP5 gene means a gene encoding an Sp5 transcription factor.
  • the gene is known to be involved in Wnt-mediated beta catenin signaling and regulation of target gene transcription.
  • ZIC4 gene refers to a gene encoding a zic family member 4 (ZIC4) protein, specifically, a zic family member protein of a C2H2-type zinc finger protein.
  • the protein encoded by the gene is known to be associated with X-linked visceral heterotaxy and holoprosencephaly type 5.
  • ENPEP gene, used herein, refers to a gene encoding a glutamyl aminopeptidase. ENPEP is known to be associated with choriocarcinoma and gestational choriocarcinoma.
  • PITX2 gene refers to a gene encoding a protein also known as paired-like homeodomain transcription factor 2, or pituitary homeobox 2. Mutations in the gene are known to be associated with Axenfeld-Rieger syndrome, and iridogoniodysgenesis syndrome.
  • SH3BP2 gene, used herein, refers to a gene encoding SH3 domain-binding protein 2 (SH3BP2) derived from a gene located on chromosome 4. The protein encoded by this gene is known to be associated with cherubism.
  • SEPP1 gene refers to a gene encoding selenoprotein P.
  • the selenoprotein is an extracellular glycoprotein, which is uncommon in that it contains 9 Sec residues per polypeptide, and is known to act as an antioxidant in the extracellular space.
  • FLJ32255 gene is an uncharacterized LOC643977, which is an RNA gene associated with the lncRNA class.
  • SHROOM1 gene, used herein, refers to a gene encoding a SHROOM family member 1 (SHROOM1) protein, which plays an important role in the development of the nervous system and other tissues and is involved in microtubule structure during cell elongation. Among the symptoms of Down syndrome, it is involved in congenital heart defects and arthritis.
  • LINC00574 gene means a long intergenic non-protein coding RNA 574, which is an RNA gene associated with the lncRNA class.
  • the LINC00574 gene is known to be associated with breast cancer.
  • LOC154449 gene is an uncharacterized LOC154449, which is an RNA gene associated with the lncRNA class.
  • PRRT4 proline rich transmembrane protein 4
  • TMEM176B transmembrane protein 176B
  • TMEM176B transmembrane protein 176B
  • MNX1 gene refers to a gene encoding a protein also known as motor neuron and pancreas homeobox 1 (MNX1) protein or homeobox HB9 (HLXB9). Mutations in the gene are known to be associated with Currarino syndrome.
  • LOC101928483 gene refers to a non-coding RNA (ncRNA) and is also referred to as a NOTCH1 associated lncRNA in T cell acute lymphoblastic leukemia 1 (NALT1) gene.
  • EGFL7 gene, used herein, refers to a gene encoding EGF-like domain-containing protein 7. Expression of the gene is endothelial cell-specific under physiological conditions, but the gene is known to be aberrantly expressed by tumor cells in human cancer.
  • NACC2 gene means a gene encoding an NACC family member 2 protein.
  • the protein encoded by the gene is known to be associated with lateral myocardial infarction and interstitial myocarditis.
  • C9orf69 gene encoding transmembrane protein 250, and is also called TMEM250. It is known that the protein encoded by the gene is capable of playing an important role in cell proliferation by promoting progression to the S phase in the cell cycle.
  • TLX1 T-cell leukemia homeobox protein 1
  • HOX11 T-cell leukemia homeobox protein 1
  • the protein encoded by the gene is known to interact with serine/threonine-protein phosphatase PP1-gamma catalytic subunit (PPP1CC), serine/threonine-protein phosphatase 2A catalytic subunit beta isoform (PPP2CB), and serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform (PPP2CA).
  • PPP1CC serine/threonine-protein phosphatase PP1-gamma catalytic subunit
  • PPP2CB serine/threonine-protein phosphatase 2A catalytic subunit beta isoform
  • PPP2CA serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform
  • FGF8 gene refers to a gene encoding a fibroblast growth factor 8 (FGF8) protein.
  • FGF8 fibroblast growth factor 8
  • the protein encoded by the gene supports androgen- and anchorage-independent growth of mammary tumor cells, and overexpression of this gene is known to increase tumor growth and angiogenesis.
  • TACC2 refers to a gene encoding transforming acidic coiled-coil-containing protein 2 (TACC2).
  • TACC2 transforming acidic coiled-coil-containing protein 2
  • the gene encodes a protein that is accumulated at the centrosome throughout the cell cycle, and the gene is present in chromosomal regions associated with tumorigenesis. Expression of the gene is known to affect progression of breast tumors.
  • CPXM2 gene, used herein, refers to a gene encoding a carboxypeptidase X, M14 family member 2 (CPXM2) protein.
  • NKX6-2 gene, used herein, refers to a gene encoding an NK6 homeobox 2 (NKX6-2) protein.
  • the protein encoded by the gene is known to be associated with spastic ataxia and autosomal recessive disease.
  • TLX1NB TLX1 Neighbor RNA gene and belongs to the lncRNA class.
  • IQSEC3 refers to a human gene known as IQ motif and Sec7 domain 3, and is also called KIAA1110. It is known that the gene is highly expressed in the brain, particularly in the amygdala, and plays an important role in learning.
  • PCDH8 gene, used herein, refers to a gene encoding a protocadherin-8 (PCDH8) protein.
  • the gene encodes an endogenous membrane protein that is thought to function in cell adhesion in a central nervous system (CNS)-specific manner.
  • CNS central nervous system
  • F7 gene refers to a gene encoding coagulation factor VII, a vitamin K-dependent factor essential for hemostasis.
  • the gene is known to be associated with factor VII deficiency and myocardial infarction.
  • SOX9 gene means a gene encoding the transcription factor SOX-9 protein. Mutations in this gene are known to be associated with skeletal malformation syndrome and campomelic dysplasia.
  • PPMAL2 gene, used herein, refers to a gene encoding a PNMA family member 8B protein, and an important paralog of the gene is PNMA8A.
  • the protein encoded by the gene is paraneoplastic antigen-like protein 8B.
  • THBD thrombomodulin gene
  • the term “THBD” gene refers to a gene encoding a thrombomodulin protein.
  • the protein encoded by the gene is a protein derived from endothelial cells of blood vessels and serves to prevent generation of blood clots, in cooperation with other factors.
  • MAPK8IP2 gene, used herein, refers to a gene encoding C-jun-amino-terminal kinase-interacting protein 2, and is also called islet-brain-2 (IB2). It is known that the protein encoded by the gene is highly expressed in the brain and is almost always lacking in Phelan-McDermid syndrome.
  • KLHDC7B refers to a gene encoding a kelch domain containing 7B protein. This gene is known to be associated with chlamydia pneumonia.
  • GPR143 gene refers to a gene encoding a G-protein coupled receptor 143 (GPR143) protein.
  • the gene is known to be regulated by microphthalmia-associated transcription factors.
  • IGHMBP2 refers a gene that encodes immunoglobulin helicase ⁇ -binding protein 2 (IGHMBP2), cardiac transcription factor 1 (CATF1), or a protein known as DNA-binding protein SMUBP-2. Mutations in the gene are known to cause distal spinal muscular atrophy type 1.
  • MRGPRD MRGPRD gene
  • the term “MRGPRD” gene refers to a gene encoding a Mas-related G-protein coupled receptor member D protein.
  • the gene is known to be associated with femoral cancer and liver rhabdomyosarcoma.
  • the presence or risk of Down syndrome in the fetus may be determined.
  • the CHODL, NCAM2, CYYR1, GRIK1, OLIG2, CLIC6, SIM2, HLCS, MX2, MX1, TMPRSS2, SLC37A1, PDE9A, CBS, CRYAA, C21orf2, TRPM2, TSPEAR, LINC00162, SSR4P1, SLC19A1, LOC100129027, MCM3AP, YBEY, and PRMT genes may be hyper-methylated in Down syndrome fetal placentas compared to blood of mothers pregnant with normal fetuses or normal fetal placentas; and the ITSN1 gene may be hypo-methylated.
  • the MXRA8, MIB2, KIF26B, SP5, ZIC4, ENPEP, PITX2, SH3BP2, SEPP1, FLJ32255, SHROOM1, LINC00574, LOC154449, PRRT4, TMEM176B, MNX1, LOC101928483, EGFL7, NACC2, C9orf69, TLX1, FGF8, TACC2, CPXM2, NKX6-2, TLX1NB, IQSEC3, PCDH8, F7, SOX9, PNMAL2, THBD, MAPK8IP2, KLHDC7B, and GPR143 genes may be hyper-methylated in Down syndrome fetal placentas compared to blood of mothers pregnant with normal fetuses or normal fetal placentas; and the IGHMBP2, and MRGPRD genes may be hypo-methylated.
  • hypo-methylation used herein, may refer to a state in which the methylation level of the experimental group is higher than that of the control group as a result of measuring the methylation level.
  • hypo-methylation used herein, may refer to a state in which the methylation level of the experimental group is lower than that of the control group as a result of measuring the methylation level.
  • the fetus When a methylation level of the first biomarker measured in a fetus is hypo-methylated or hyper-methylated by 10% to 30% or more, compared to the methylation level measured in the blood of mothers pregnant with a normal fetus or in normal fetal placentas, the fetus may be determined to have or be at a high risk of having Down syndrome.
  • the methylation level of the first biomarker measured in normal fetal placentas was confirmed to be 10% to 20% or more hypo-methylated or hyper-methylated compared to the methylation level measured in normal maternal blood, and the first biomarker was confirmed to be a tissue (placenta)-specific marker.
  • the methylation level of the first biomarker measured in Down syndrome fetal placentas was confirmed to be 10% to 20% or more hypo-methylated or hyper-methylated compared to the methylation level measured in normal fetal placentas, and the first biomarker was confirmed to be a disease (Down syndrome)-specific marker.
  • the fetus When a methylation level of the second biomarker measured in a fetus is hypo-methylated or hyper-methylated by 30% to 50% or more, compared to the methylation level measured in the blood of mothers pregnant with a normal fetus or in normal fetal placentas, the fetus may be determined to have or be at a high risk of having Down syndrome.
  • the methylation level of the first biomarker measured in normal fetal placentas was confirmed to be 10% to 20% or more hypo-methylated or hyper-methylated compared to the methylation level measured in normal maternal blood, and the second biomarker was confirmed to be a tissue (placenta)-specific marker.
  • the methylation level of the second biomarker measured in Down syndrome fetal placentas was confirmed to be 30% to 50% or more hypo-methylated or hyper-methylated compared to the methylation level measured in normal fetal placentas, and the first biomarker was confirmed to be a disease (Down syndrome)-specific marker.
  • biological sample may include samples such as tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, and urine isolated from a fetus, and may include cell-free DNA, which is DNA free in the blood and is not present in the cell nucleus.
  • the biological sample may be derived from the placenta.
  • placenta refers to a structure that mediates material exchange between a fetus and the mother necessary for the growth and survival of the fetus, and is formed when a part of the fetal membrane surrounding the fetus adheres to the mother's endometrium.
  • the placenta may include chorion.
  • chorion corresponds to the middle layer among the decidua, chorion, and amnion, which are membranes that enclose amniotic fluid in the uterus.
  • the biological sample may be derived from chorionic villus cells.
  • the biological sample may refer to cell-free DNA in maternal blood derived from chorionic villus cells.
  • Another aspect provides a method of diagnosing Down syndrome including: measuring a methylation level of a Down syndrome biomarker in a biological sample separated from a subject, wherein the biomarker is a first biomarker present on chromosome 21, a second biomarker present on a chromosome other than chromosome 21, or a combination thereof; comparing the measured methylation level of the biomarker with a methylation level of the first biomarker and the second biomarker in the biological sample separated from a normal control group; and determining a presence or a risk of Down syndrome by comparing the methylation levels.
  • the method according to an aspect has an effect of efficiently diagnosing Down syndrome by simple measurement of gene methylation levels.
  • a normal control group for example, blood of mothers pregnant with a normal fetus or normal fetal placentas
  • a presence or risk of Down syndrome in the fetus may be diagnosed.
  • FIG. 1 shows results of comparing average methylation levels among 65 biomarker groups through analysis of methylation levels of chromosome 21 for normal maternal blood (NB), normal fetal placental tissue (NT), and Down syndrome fetal placental tissue (T21T).
  • FIG. 2 shows results of comparing methylation levels among 33 biomarker groups through analysis of methylation levels of a genome for the normal maternal blood (NB), the normal fetal placental tissue (NT), and the Down syndrome fetal placental tissue (T21T).
  • the placental tissue was chorionic villus cells used for chorionic villus tests which are harvested in early pregnancy (12 to 13 weeks of gestation) and stored in liquid nitrogen until analysis. The gestational age of each fetus was determined by ultrasonography. A chromosome analysis using the Giemsa-trypsin-Giemsa (GTG) banding method was performed to determine the karyotype of fetal chorionic villus cells.
  • GCG Giemsa-trypsin-Giemsa
  • DNA methylation was quantified at various CpG sites by using MC-seq, and methylome profiling was performed.
  • a standard DNA methylation region capture library was generated by using the SureSelect Methyl-Seq Target Enrichment protocol (Agilent) for paired-end sequencing libraries (ver. B.3, June 2015; Illumina) with 3 ⁇ g of genomic DNA.
  • a SureSelect Human Methyl-Seq probe set (Agilent, catalog number 5190-4662) was used. Quantification of DNA and quality assessment of DNA were performed by measuring with a PicoGreen assay kit (Thermo Fisher Scientific, catalog number P7589) and a Nanodrop spectrophotometer (NanoDrop Technologies, catalog number ND-2000), respectively.
  • 3 ⁇ g of the genome was fragmented to a target size of 150 bp to 200 bp of DNA by using an ultrasonicator (AFA; Covaris, catalog number 500219). Briefly, 8 microtube strips were loaded onto the tube holder of the sonicator and DNA was sheared by using the following setting: mode, frequency sweeping; duty cycle, 10%; intensity, 5; cycles per burst, 200; duration, 60 sec ⁇ 6 cycles; temperature, 4° C. to 7° C. The fragmented DNAs were repaired, ‘A’ was ligated to the 3′ end, and SureSelect Methyl-Seq Methylated Adapter was ligated to the fragment. After the ligation was evaluated, the adapter-ligated products were amplified by PCR.
  • AFA Ultrasonicator
  • the final purified product such as methylated adapter-ligated DNA was then quantified according to the qPCR quantification protocol guide and verified by using a TapeStation DNA screen tape D1000 (Agilent, catalog number 5067-5582).
  • DNA methylation region capture 350 ng of DNA library was mixed with hybridization buffer, blocking mix, RNase block, and 5 ⁇ l of SureSelect All DNA methylation region capture library according to the standard SureSelect Methyl-Seq Target Enrichment protocol (Agilent).
  • Hybridization with capture baits was performed at 65° C. by using a PCR machine with a thermal cycler read option heated at 105° C. for 24 hours.
  • the target captured DNA was treated with bisulfite by using a EZ DNA Methylation-Gold Kit (Zymo Research, catalog number D5005), and 8 PCR cycles to enrich the adapter-added fragments and 6 PCR cycles to add multiplexing barcodes were performed.
  • the captured DNA was amplified.
  • the final purified product was quantified by using the qPCR quantification protocol guide mentioned above and verified by using the TapeStation DNA screen tape D1000 (Agilent, catalog number 5067-582). Sequencing was performed by using the HiSeqTM 2500 platform (Illumina, catalog number SY-401-2501).
  • the washed reads were aligned to the Homo sapiens genome (UCSC hg19) by using a bisulfite sequencing MAPping program (BSMAP; version 2.90 parameters set-n 1 -r 0) based on an unidirectional short oligonucleotide alignment program (SOAP), and the washed reads could be uniquely mapped in the data.
  • BSMAP bisulfite sequencing MAPping program
  • SOAP unidirectional short oligonucleotide alignment program
  • a methylation ratio higher than the 10 CT number of all single cytosines located in the Agilent SureSelect target region may indicate that general methylation is completed. For regions covered by both ends of a read pair, only one read was used to call methylation.
  • the profiles within the range the results are applied were summarized as follows: # of C/actual CT number for each of the three sequence contexts (CG, CHG, and CHH).
  • the methylation level at each base of CpG was normalized with intermediate scaling normalization to distinguish between DMCs and DMRs.
  • an independent T-test was used to assess significance of differences of the methylation between the two groups.
  • false discovery results were controlled in multiple tests by using the Benjamini and Hochberg false discovery rate (FDR) method, and correcting.
  • FDR Benjamini and Hochberg false discovery rate
  • DMR was defined as a contiguous region of any length containing ⁇ 3 DMCs.
  • a hierarchical clustering analysis was also performed by using complete linkage and Euclidean distance as measures of similarity for indicating methylation levels of samples for significant CpGs that satisfy one or more comparison pairs. Heatmaps were automatically plotted by centroid linkage by using the centroid absolute correlation of similarity metric. All data analysis and visualization of differentially methylated results were performed by using R 3.3.1 (www.r-project.org) and Statistical Package for Social Sciences 12.0 (SPSS Inc.).
  • Genomic methylation patterns were comparatively analyzed at a CpG site level by using the method of Example 2, for 5 samples each of blood of mothers with normal fetuses, normal fetal placentas, and Down syndrome fetal placentas obtained in Example 1.
  • the methylation level (%) of each CpG site was expressed in a scale of 0 to 100, with 0 being unmethylated and 100 being completely methylated.
  • hyper-methylated or hypo-methylated biomarkers were discovered.
  • the CHODL, NCAM2, CYYR1, GRIK1, OLIG2, CLIC6, SIM2, HLCS, MX2, MX1, TMPRSS2, SLC37A1, PDE9A, CBS, CRYAA, TRPM2, C21orf2, TSPEAR, LINC00162, SSR4P1, SLC19A1, LOC100129027, MCM3AP, YBEY, PRMT2, and ITSN1 gene regions were selected as regions with three or more consecutive epigenetic characteristics of the same type from chromosome 21, the target chromosome of Down syndrome. Detailed information of the selected 65 gene regions is shown in Table 4 below.
  • the degrees of methylation (the value obtained by dividing the methylation level (%) by 100) of the selected gene regions in normal maternal blood, normal fetal placentas, and Down syndrome fetal placentas are shown in Table 5 below and FIG. 1 .
  • a difference in methylation levels between normal fetal placentas and maternal blood cells, and a difference in methylation levels between Down syndrome fetal placentas and normal fetal placentas were compared.
  • the CHODL, NCAM2, CYYR1, GRIK1, OLIG2, CLIC6, SIM2, HLCS, MX2, MX1, TMPRSS2, SLC37A1, PDE9A, CBS, CRYAA, TRPM2, C21orf2, TSPEAR, SSR4P1, LINC00162, MCM3AP, YBEY, and PRMT2 (NONE) gene regions were confirmed to be hyper-methylated in the fetal placentas, especially in the Down syndrome fetal placentas.
  • the difference in methylation levels of the genes between the normal fetal placentas and maternal blood cells was 10 to 55, and the genes were hyper-methylated in the fetal placentas compared to the maternal blood, and thus, the genes were confirmed to be tissue (placenta)-specific biomarkers.
  • the difference in methylation levels of the genes between the Down syndrome fetal placentas and normal fetal placentas was 10 to 20, confirming that the genes are disease (Down syndrome)-specific biomarkers hyper-methylated in a Down syndrome fetus compared to a normal fetus. Differences in the methylation levels among the Down syndrome fetal placentas and the other two groups (normal fetal placentas and maternal blood) were all statistically significant (P ⁇ 0.05).
  • the ITSN1 gene region was hypo-methylated in the Down syndrome fetal placentas. Specifically, it was confirmed that the methylation level of the ITSN1 gene region in maternal blood cells was 90 or more, and the methylation level in the normal fetal placentas was 65 or more, and hyper-methylated, whereas the methylation level in the Down syndrome fetal placentas was 40 or less, and hypo-methylated. The difference in methylation levels between the groups was 25 or more. Even in this case, differences in the methylation levels among the Down syndrome fetal placentas and the other two groups (normal fetal placentas and maternal blood) were all statistically significant (P ⁇ 0.05).
  • the MXRA8, MIB2, KIF26B, SP5, ZIC4, ENPEP, PITX2, SH3BP2, SEPP1, FLJ32255, SHROOM1, LINC00574, LOC154449, PRRT4, TMEM176B, MNX1, LOC101928483, EGFL7, NACC2, C9orf69, TLX1, FGF8, TACC2, CPXM2, NKX6-2, TLX1NB, IQSEC3, PCDH8, F7, SOX9, PNMAL2, THBD, MAPK8IP2, KLHDC7B, GPR143, IGHMBP2, and MRGPRD gene regions were selected as regions with two or more consecutive epigenetic characteristics of the same type. Detailed information of the selected 33 gene regions is shown in Table 6 below.
  • the degrees of methylation (the value obtained by dividing the methylation level (%) by 100) of the selected gene regions in the normal maternal blood, normal fetal placentas, and Down syndrome fetal placentas are shown in Table 7 below and FIG. 2 .
  • a difference in methylation levels between the normal fetal placentas and maternal blood cells, and a difference in methylation levels between the Down syndrome fetal placentas and normal fetal placentas were compared.
  • the difference in methylation levels of the genes between the normal fetal placentas and maternal blood cells was 10 to 50, and the genes were hyper-methylated in the fetal placentas compared to the maternal blood, and thus, the genes were confirmed to be tissue (placenta)-specific biomarkers.
  • the difference in methylation levels of the genes between the Down syndrome fetal placentas and normal fetal placentas was 25 to 50, confirming that the genes are disease (Down syndrome)-specific biomarkers hyper-methylated in a Down syndrome fetus compared to a normal fetus. Differences in the methylation levels among the Down syndrome fetal placentas and the other two groups (normal fetal placentas and maternal blood) were all statistically significant (P ⁇ 0.05).
  • the IGHMBP2 and MRGPRD gene regions were hypo-methylated in the Down syndrome fetal placentas. It was confirmed that the methylation levels of the IGHMBP2 and MRGPRD gene regions in normal fetal placentas and maternal blood cells were 75 or more, and hyper-methylated, whereas the methylation levels in Down syndrome fetal placentas were 15 or less, and hypo-methylated. Even in this case, differences in the methylation levels among the Down syndrome fetal placentas and the other two groups (normal fetal placentas and maternal blood) were all statistically significant (P ⁇ 0.05).
  • Example 4 Summarizing the results of Example 4, it may be confirmed that the genes MXRA8, MIB2, KIF26B, SP5, ZIC4, ENPEP, PITX2, SH3BP2, SEPP1, FLJ32255, SHROOM1, LINC00574, LOC154449, PRRT4, TMEM176B, MNX1, EGFL7, LOC101928483, NACC2, C9orf69, TLX1, FGF8, TACC2, CPXM2, NKX6-2, TLX1NB, IQSEC3, PCDH8, F7, SOX9, PNMAL2, THBD, MAPK8IP2, KLHDC7B, and GPR143; and CHODL, NCAM2, CYYR1, GRIK1, OLIG2, CLIC6, SIM2, HLCS, MX2, MX1, TMPRSS2, SLC37A1, PDE9A, CBS, CRYAA, C21orf2, TRPM2, TSPEAR, LINC00162, SSR4P1, SLC19A1,

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